Central to an efficient adaptive immune response is the ability to tightly regulate immune activation, to avoid both pathology to self-antigens and collateral damage during infectious challenges. Multiple mechanisms exist to control immune activation. Regulatory T cells (CD4+CD25+, Tregs), a distinct subset of CD4+ T cells, are a critical immune-modulating cell type for responses to self and foreign antigen. Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that results in the destruction of the insulinproducing (3 cells of the pancreas. We have made a number of observations in humans that give us a novel handle on immune regulation in recent onset juvenile diabetics. CD4+CD25+ Treg activity is significantly impaired at diabetes diagnosis in a subset of subjects. In a longitudinal study, preliminary data suggests that CD4+CD25+ Treg activity declines from diagnosis. Using multi-chromatic flow cytometry we have found a striking heterogeneity in the CD25+ comrpatment that differs between diabetics and control. Based on these data, Specific aim 1 tests the hypothesis that diabetes is precipitated by a confluence of increased number or function of anti-islet effector cells and decreased Treg function. Given the phenotypic heterogeneity within this subset we will determine if the changes in function are due to defects in classic Treg activity or due to the emergence of a circulating CD4+Foxp3+ pathogenic pool. Subsets will be sorted based on phenotype and specificity (anti-islet antigen tetramers) and function determined directly ex vivo using single cell analyses. Our studies in the NOD mouse have revealed phenotypic differences in Foxp3+ T cells between the pancreatic lymph node and the pancreas suggesting local Treg modulation. Specific Aim 2, tests the hypothesis that the diabetic pancreas is hostile for Treg function. It follows that changes in the pancreatic infiltrate from respectful to destructive insulitis may trigger the local loss of Treg activity propagating tissue demise. We will utilize Foxp3-GFP reporter NOD and BDC2.2.NOD mouse strains to characterize the changes in Treg phenotype, function and location in the pancreatic tissue.